Tuberculosis (TB) is caused by infection with the bacterium Mycobacteria tuberculosis (Mtb) and is the leading cause of mortality in the world for a single infectious agent. As efforts to treat TB expand, the prevalence of multidrug resistant TB (MDR-TB), which are resistant to the frontline standard of care (SOC) antibiotics rifampicin and isoniazid, is increasing. Despite the dire need for new treatments against drug resistant TB, only one new class of antibiotics has made it into the clinic for treatment of MDR-TB in the past 40 years and the approved drug from this class, bedaquiline, has significant side effects, including death. Therefore, new classes of drugs that target Mtb in ways that synergize with existing drug sensitive TB and MDR-TB SOC therapies are desperately needed. To this end, Fimbrion has in-licensed the intellectual property for a series of heterocyclic compounds with inhibitory and bactericidal activity against Mtb in vitro. This technology originated at Washington University and Saint Louis University, and the most potent members of these series have activity at inhibitor concentrations (IC50) in the low nanomolar range. The target of this compound series is the QcrB protein, a component of the respiratory electron transport chain in Mtb, suggesting that these compounds inhibit Mtb growth and survival through disrupting respiration. QcrB has recently been identified as a viable drug target for treating TB, but our compound series has a unique structure compared to the one QcrB inhibitor, Q203, that is currently in clinical trials and our compounds have bactericidal activity in vitro, whereas bactericidal activity has not been reported for Q203. While many physiochemical properties of this scaffold are suitable for drug development and we have generated some very potent compounds, low metabolic stability has thus far been a liability. Therefore, optimizing stability, while maintaining potency, will be a priority of our chemical optimization efforts. The main goal or our project proposal is to develop a lead series of novel heterocyclic QcrB inhibitors with improved pharmacokinetic (PK) properties that will be capable of effectively treating TB in an animal model of Mtb infection. To achieve this goal, we will expand our library of compounds, focusing on two subclasses of heterocycles in order to discover and optimize candidate lead series with increased metabolic stability, while maintaining potency and minimizing cellular toxicity. To properly direct lead series identification and optimization, we will select early lead compounds initially and then later optimized lead series compounds with high potency and increased stability for in vivo PK. Candidate leads with favorable PK profiles will be tested for in vitro activity against a collection of 10 diverse drug-sensitive and MDR Mtb strains as well as for synergy with bedaquiline, which also targets Mtb respiration. We will then perform proof of principle experiments to test whether an advanced lead series compound can have in vivo efficacy in an animal model of acute Mtb lung infection. The proposed studies have the potential to result in the development of a new family of anti-mycobacterials for use against both drug-sensitive and drug-resistant TB.